JP2015521054A5 - - Google Patents

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JP2015521054A5
JP2015521054A5 JP2015516082A JP2015516082A JP2015521054A5 JP 2015521054 A5 JP2015521054 A5 JP 2015521054A5 JP 2015516082 A JP2015516082 A JP 2015516082A JP 2015516082 A JP2015516082 A JP 2015516082A JP 2015521054 A5 JP2015521054 A5 JP 2015521054A5
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cdc
explant
cells
stem cell
cell therapy
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JP2015516082A
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Priority claimed from PCT/US2013/043772 external-priority patent/WO2013184527A1/en
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Claims (23)

同種異系心臓幹細胞療法に適切な、ろ過された心筋球由来細胞（ＣＤＣ）の調製方法であって、
対象から受け取ったドナー心臓組織の小片を、約２００μｍ3から約６００μｍ3の範囲の複数の組織外植片に処理し、前記外植片が約０．３から約０．６グラムの範囲であるステップ；
前記外植片を、約３０秒から約５分の範囲の時間、酵素的に消化するステップ；
前記外植片を、約１外植片／４００から７００ｃｍ2培養表面積の密度で、細胞が前記外植片から遊走するまで培養するステップ；
外植片から遊走する細胞を収集するステップ；
収集した細胞を、ＣＤＣを作製するために培養するステップ；
ＣＤＣを採取するステップ；および
前記採取されたＣＤＣを、直径５０μｍより大きい粒子を除去して、直径５０μｍ未満のＣＤＣを単離して、同種異系心臓幹細胞療法を容易にするためにろ過するステップ；
を含む方法。 A method of preparing filtered cardiomyocyte-derived cells (CDC) suitable for allogeneic cardiac stem cell therapy comprising:
A piece of donor heart tissue received from a subject is processed into a plurality of tissue explants ranging from about 200 μm 3 to about 600 μm 3 , the explant ranging from about 0.3 to about 0.6 grams. Step;
Enzymatically digesting the explant for a time ranging from about 30 seconds to about 5 minutes;
Culturing the explant at a density of about 1 explant / 400 to 700 cm 2 culture surface area until cells migrate from the explant;
Collecting cells that migrate from the explant;
Culturing the collected cells to produce CDC;
Collecting the CDC; and filtering the collected CDC to remove particles larger than 50 μm in diameter and isolate CDC less than 50 μm in diameter to facilitate allogeneic cardiac stem cell therapy;
Including methods. 直径５０μｍ未満にサイズ分けされた単離ＣＤＣが、冠動脈内送達および内径約５０μｍを有する心臓細動脈への通過に適切である、請求項１に記載の方法。   The method of claim 1, wherein isolated CDC sized less than 50 μm in diameter is suitable for intracoronary delivery and passage to cardiac arterioles having an inner diameter of about 50 μm. 前記ろ過ステップが、前記採取されたＣＤＣを、第１のフィルターを介してろ過し、約１４０μｍより大きい粒子を除去するステップおよび第２のフィルターを介してろ過し、約５０μｍより大きい粒子を除去するステップを含む、請求項１に記載の方法。   The filtration step filters the collected CDC through a first filter to remove particles larger than about 140 μm and a second filter to remove particles larger than about 50 μm. The method of claim 1, comprising steps. 前記第１および前記第２のフィルターが、単一の２段階フィルター装置を構成する、請求項３に記載の方法。   The method of claim 3, wherein the first and second filters constitute a single two-stage filter device. 前記ドナー心臓組織が、前記処理の前に、約１から約９０日の範囲の期間、場合により凍結保存される、請求項１に記載の方法。   The method of claim 1, wherein the donor heart tissue is optionally cryopreserved for a period ranging from about 1 to about 90 days prior to the treatment. 前記処理ステップが、培養容器中の外植片を手動で配置するのではなく、外植片を培養培地であふれさせることによって、前記外植片を解剖容器から培養容器に移動させるステップをさらに含み、前記あふれさせることによる移動が、汚染の危険性を低下させ、前記外植片の攪乱を低下させる、請求項１に記載の方法。   The processing step further includes the step of moving the explant from the dissection container to the culture container by overflowing the explant with a culture medium instead of manually placing the explant in the culture container. The method of claim 1, wherein movement by overflowing reduces a risk of contamination and reduces disturbance of the explant. 前記複数の外植片が、ドナー心臓組織の１つの領域から得られた複数の外植片を含む、請求項１に記載の方法。   The method of claim 1, wherein the plurality of explants comprises a plurality of explants obtained from a region of donor heart tissue. 前記複数の外植片が、ドナー心臓組織の複数の領域から得られた複数の外植片を含む、請求項１に記載の方法。   The method of claim 1, wherein the plurality of explants comprises a plurality of explants obtained from a plurality of regions of donor heart tissue. 前記培養ステップが、前記外植片から遊走する細胞の凝集を減少させるために、ヘパリンおよびＬ−グルタミンの１つ以上を培養培地に加えるステップをさらに含む、請求項１に記載の方法。   The method of claim 1, wherein the culturing step further comprises adding one or more of heparin and L-glutamine to the culture medium to reduce aggregation of cells migrating from the explant. 同種異系心臓幹細胞療法に適切なＣＤＣを凍結するステップをさらに含む、請求項１に記載の方法。   2. The method of claim 1, further comprising the step of freezing the CDC suitable for allogeneic cardiac stem cell therapy. ドナー心臓組織が、約１グラムから約３００グラムのサイズに及ぶ、請求項１に記載の方法。   The method of claim 1, wherein the donor heart tissue ranges in size from about 1 gram to about 300 grams. 前記処理ステップが、立方体形状である複数の組織外植片をもたらす、請求項１に記載の方法。   The method of claim 1, wherein the processing step results in a plurality of tissue explants that are cube-shaped. ドナー心臓組織が、前記対象から摘出されてから３日以内に処理される、請求項１に記載の方法。   The method of claim 1, wherein donor heart tissue is processed within 3 days of being removed from the subject. 同種異系心臓幹細胞療法に適切な前記ＣＤＣが、１４０μｍより大きい粒子を１％未満含む、請求項１から１３のいずれか一項に記載の方法。   14. The method of any one of claims 1 to 13, wherein the CDC suitable for allogeneic cardiac stem cell therapy comprises less than 1% particles greater than 140 [mu] m. 同種異系心臓幹細胞療法に適切な前記ＣＤＣが、１５０μｍより大きい粒子を実質的に０％含む、請求項１から１３のいずれか一項に記載の方法。   14. A method according to any one of claims 1 to 13, wherein the CDC suitable for allogeneic cardiac stem cell therapy comprises substantially 0% of particles greater than 150 [mu] m. 同種異系心臓幹細胞療法に適切な前記ＣＤＣが、５０μｍより大きい粒子を１％未満含む、請求項１から１３のいずれか一項に記載の方法。   14. The method of any one of claims 1 to 13, wherein the CDC suitable for allogeneic cardiac stem cell therapy comprises less than 1% particles greater than 50 [mu] m. 前記単離ＣＤＣは、冠動脈内送達により対象に送達されるためのものである、請求項１から１３のいずれか一項に記載の方法。 14. The method of any one of claims 1 to 13, wherein the isolated CDC is for delivery to a subject by intracoronary delivery . 請求項１から１３のいずれか一項に記載の方法に従って作製された単離ＣＤＣ。   An isolated CDC made according to the method of any one of claims 1-13. 同種異系心臓幹細胞療法に適切な心筋球由来細胞（ＣＤＣ）を調製する方法であって、
第１の対象から受け取ったドナー心臓組織の小片を、約０．３から約０．６グラムの範囲の複数の立方体状組織外植片に処理するステップ；
前記外植片を、約１外植片／４００から７００ｃｍ2培養表面積の密度で、細胞が前記外植片から遊走するまで培養するステップ；
外植片から遊走する細胞を収集するステップ；
収集した細胞を、ＣＤＣを作製するために培養するステップ；
ＣＤＣを採取するステップ；
前記採取されたＣＤＣをろ過し、約５０μｍより大きい粒子を除去して、それによって、同種異系心臓幹細胞療法に適切なＣＤＣを作製するステップ；
を含む方法。 A method for preparing cardiomyocyte-derived cells (CDC) suitable for allogeneic cardiac stem cell therapy comprising:
Processing a piece of donor heart tissue received from a first subject into a plurality of cubic tissue explants ranging from about 0.3 to about 0.6 grams;
Culturing the explant at a density of about 1 explant / 400 to 700 cm 2 culture surface area until cells migrate from the explant;
Collecting cells that migrate from the explant;
Culturing the collected cells to produce CDC;
Collecting CDC;
Filtering the harvested CDC to remove particles greater than about 50 μm, thereby creating a CDC suitable for allogeneic cardiac stem cell therapy;
Including methods. 同種異系心臓幹細胞療法に適切な心筋球由来細胞（ＣＤＣ）を含む組成物であって：
第１の対象由来のドナー心臓組織から作製された複数のＣＤＣ；
１％未満の１４０μｍより大きいサイズの粒子；
１％未満の５０μｍより大きいサイズの粒子、
を含み、
前記組成物が、冠動脈内送達経路による対象への投与に適切である組成物。 A composition comprising cardiomyocyte-derived cells (CDC) suitable for allogeneic cardiac stem cell therapy comprising:
A plurality of CDCs made from donor heart tissue from a first subject;
Less than 1% of particles larger than 140 μm;
Particles less than 1% in size larger than 50 μm,
Including
A composition wherein the composition is suitable for administration to a subject by an intracoronary delivery route. 前記組成物が、ＣＤＣの実質的に純粋な集団を含む、請求項２０に記載の組成物。   21. The composition of claim 20, wherein the composition comprises a substantially pure population of CDC. 前記組成物が、実質的に均一なサイズのＣＤＣ集団を含み、全ＣＤＣの９８％より多くが４０μｍ未満である、請求項２０または２１のいずれか一項に記載の組成物。   22. The composition according to any one of claims 20 or 21, wherein the composition comprises a substantially uniform sized CDC population and more than 98% of the total CDC is less than 40 [mu] m. 前記外植片から遊走する細胞を収集するステップの後、および、前記収集した細胞を、ＣＤＣを作製するために培養するステップの前に、前記外植片から遊走する細胞をマスターセルバンクとして凍結保存するステップを更に含む、請求項１から１３のいずれか一項に記載の方法。  After the step of collecting cells that migrate from the explant and before the step of culturing the collected cells to produce CDC, the cells that migrate from the explant are cryopreserved as a master cell bank. The method according to claim 1, further comprising the step of:
JP2015516082A 2012-06-05 2013-05-31 Optimized methods for generating cardiac stem cells from heart tissue and their use in cardiac therapy Pending JP2015521054A (en)

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